Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the beta and common gamma chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the alpha chain of the IL-15 receptor (IL-15Ralpha). We have shown that membrane-associated IL-15RalphaIL-15 complexes were transferred from presenting cells to NK cells through trans-endocytosis and stimulated efficient phosphorylation of ribosomal protein S6 and NK cell proliferation. Conversely, NK cell interaction with soluble or surface-bound IL-15RalphaIL-15 complex resulted in preferential Stat5 phosphorylation and NK cell survival. Inhibition of metalloprotease-induced IL-15RalphaIL-15 shedding from trans-presenting cells reduced Stat5 phosphorylation in NK cells but had no effect on S6 phosphorylation. Conversely, inhibition of trans-endocytosis by silencing the small GTPase TC21, or by expression of a dominant-negative TC21, reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15RalphaIL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment where receptorligand interaction occurs can dictate functional outcome. They have also important implications for the stimulation and use of NK cells in cancer immunotherapy. NK cells are an important component of the immune system, contributing to both pathogen defense and tumor immune surveillance. Clinical data has shown a correlation of higher NK cell infiltration into tumors with better immunotherapy outcome. Unlike T and B cells, which use a large repertoire of receptors to achieve antigen specificity, NK cell activity is regulated by a limited number of germline-encoded activating and inhibitory receptors. Activation of resting NK cells requires synergy of specific pairs of activating receptors. The CD28B7 family of receptorligand pairs regulates T cell responses through costimulation and coinhibition. It includes checkpoint inhibitors, such as PD-1, which limit anti-tumor responses. CD28 homologue (CD28H, encoded by the TMIGD2 gene) has been reported as a costimulator and coinhibitor of T cells. We have shown that CD28H is a strong coactivation receptor on NK cells that synergizes with receptors 2B4 and NKp46 to induce NK cell degranulation and lysis of target cells. CD28H binding to its ligand B7-homologue 7 (B7H7, encoded by the HHLA2 gene) enhanced antibody-dependent cellular cytotoxicity (ADCC) of NK cells. NK-dependent lysis of a B7H7+ tumor cell line was inhibited by the dominant inhibitory receptor for HLA-E. However, expression of a chimeric antigen receptor, consisting of CD28H fused to TCR, in NK cells could overcome inhibition and triggered tumor cell lysis. Thus, CD28H is a new addition to the arsenal of activation receptors used by NK cells to detect tumor cells. Natural killer (NK) cells are innate immune effector cells regulated by many germline encoded activating and inhibitory receptors. The DAP12-associated activating receptor KIR2DS4 has been linked with multiple diseases including cancer, disorders of pregnancy, and resistance to HIV. However, the ligand(s) for KIR2DS4 have remained poorly defined, and the role of this receptor in immune responses is unclear. We have shown that human KIR2DS4 is a highly peptide-specific receptor for the human MHC-I molecule HLA-C*05:01. Of over 60 different peptides tested, only two conferred binding to KIR2DS4. Recognition of these peptides presented by HLA-C*05:01 potently activated KIR2DS4+ NK cells to degranulate and produce IFN-gamma and TNF-alpha. Recombinant peptide:HLA-C alone was sufficient to activate KIR2DS4+ NK cells and we estimated the number of HLA-C molecules required for stimulation to be in the range of only a thousand per cell. An alignment search of the KIR2DS4 binding peptides identified an epitope in recombinase A (RecA), a highly conserved bacterial protein. RecA epitopes from the pathogens Chlamydia, Campylobacter, Brucella and Helicobacter were presented by HLA-C*05:01, bound to KIR2DS4, and activated KIR2DS4+ NK cells. By sequence alignment we predict that hundreds of species of bacteria contain RecA epitopes that can be presented by HLA-C*05:01 and bind KIR2DS4. These data provide clear evidence that KIR2DS4 is a highly peptide specific activating receptor and suggest that KIR2DS4 evolved to play a role in immune defense to bacteria.